Local retention efficacy of steroid-loaded PLGA microspheres in epidural injection

Long-term effects of epidural steroid injections for pain management require novel drug formulations that increase tissue retention time. Present study aimed to investigate the local retention of steroid-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in epidural injection using a rabbit model. Twenty rabbits were randomly assigned to a PLGA group (n = 10) and a triamcinolone acetonide (TA) group (n = 10). Each animal was injected with either TA-loaded PLGA microspheres or conventional TA suspension into the lumbar epidural space. The lumbar segments were then harvested from the sacrificed rabbits on day 1, week 1, 2, and 4 after the injection. On day 1, the residual steroid concentration (RSC) was lower in the PLGA group than in the TA group (5.03 ppm vs. 13.01 ppm). However, after a week, more steroids remained in the PLGA group (3.29 ppm vs. 0.58 ppm). After 2 weeks, fewer steroids remained in the PLGA group than in the TA group, although both contained less than 10% of the initial retention dose. This study shows that steroid-loaded PLGA tended to have higher steroid retention in tissue than the steroid itself at the first week after epidural injection. However, most of the steroids disappeared after 2 weeks in both groups.


Materials and methods
The study protocols were authorized and approved by the Animal Care and Use Committee of our Institute (approval number: BA1608-206/050-01) before the commencement of the study and reported in accordance with ARRIVE guidelines. All animal experiments were performed in compliance with the Principles of Laboratory Animal Care (NIH publication number 85-23, revised 1996) and the approved guidelines. This experimental study was conducted from July 2018 to August 2019.
Animals. Twenty-five female New Zealand White rabbits weighing 3.6 ± 0.4 kg (range, 3.0-4.0 kg) were obtained from DooYeol Biotech (Seoul, Korea), and Saeronbio (Uiwang-si, Korea). The rabbits were acclimatized to the animal experimental platform for 1 week prior to the experiments. The animals were housed in individual cages under standard laboratory conditions including a controlled light cycle (12-h light/12-h dark) and controlled temperature (21 ± 2 ºC). Tap water and standard laboratory chow were provided ad libitum. We initially planned to use 20 rabbits. However, a total of 25 rabbits were used in the experiment, as 5 of them died for unknown reasons immediately after anesthesia or during transport (Fig. 1). Rabbits were randomly assigned to an experimental group (n = 10, PLGA group) or a control group (n = 10, triamcinolone acetonide (TA) group). To assess the local steroid retention in the lumbar segments over time, we sacrificed the rabbits in both groups at scheduled time intervals from the day of the epidural injection, namely, on day 1 (n = 1 for each group), and at weeks 1, 2, and 4 (n = 3 for each group and each time point).
The particle size and homogeneity of the TA-loaded PLGA microspheres were determined using a laser diffraction particle size analyzer (LA-950, Horiba Ltd., Kyoto, Japan). The TA-loaded PLGA microspheres fabricated by the spray-drying procedure were highly spherical, with a smooth and homogeneous surface (Fig. 2). The loading efficiency of TA microcrystals on PLGA microsphere was greater than 95%.
Experimental design and epidural injection. All experiments were conducted in fully anesthetized animals. Anesthesia was performed via intramuscular injection of alfaxalone (Alfaxan, 10 mg/ml; Jurox Pty, Ltd., Rutherford, Australia; 5 mg/kg body weight) and xylazine (Rompun, 23.32 mg/ml; Bayer Korea, Ansan, Korea; 5 mg/kg body weight) for all 20 rabbits before ESI. The anesthetized rabbit was placed on a fluoroscopy table in Euthanization and tissue harvesting. The tissue was harvested from the sacrificed animals at the predetermined time intervals after the epidural injection. In each group, the rabbits were anesthetized by performing consecutive intramuscular and intravenous injection of alfaxalone (Alfaxan, 10 mg/ml; Jurox Pty, Ltd., Rutherford, Australia) and xylazine (Rompun, 23.32 mg/ml; Bayer Korea, Ansan, Korea) as follows: intramuscular injection with alfaxalone (0.7 ml/kg) and xylazine (0.3 ml/kg) followed by intravenous injection with alfaxalone (0.35 ml/kg) and xylazine (0.15 ml/kg). The fully anesthetized rabbits were euthanized by intravenous injection of potassium chloride (150 mg/ml; JW Pharmaceutical, Seoul, Korea; 150 mg/kg body weight). After death was confirmed by loss of heart rate, two consecutive lumbar segments comprising the injection segment (L6/L7) and the adjacent segment (L4/L5) were harvested (Fig. 4). All tissue samples were kept separately on ice until further  Statistical analysis. Distribution normality of the data was evaluated using the Spiro-Wilk normality test. Data with normal distribution were expressed as mean and standard deviation (SD), while non-normally distributed data were presented as median and interquartile range (IQR). Experimental data were presented descriptively as percentages and plot charts. Generalized estimating equations were applied to assess the difference in the RSC between the drugs over time. Analyses were done with R software (version 4.0.2; The R Project for Statistical Computing), and P-values less than 0.05 were considered statistically significant.

Results
The morphological and physical characteristics of the TA-loaded PLGA microsphere are shown in Fig. 2. The median diameter (d50) of the TA-loaded PLGA microspheres was determined to be 2.77 μm, with a narrow size distribution (span value 2.71). The mean weight of the extracted lumbar segments was 50.0 ± 4.5 mg. The RSC values of the animals are shown in Table 1 and Fig. 5. To intuitively compare RSCs, the values were expressed as a percentage of each group's day 1 results (Table 1). Regarding the injected segment, the RSC values obtained immediately after ESI (day 1) were lower in the PLGA group than in the TA group (5.03 ± 0.16 ppm vs. 13.01 ± 0.64 ppm). In the PLGA group, the steroid remained almost completely in an animal at week 1, while about half of the steroids were detected in the other two animals at week 1 (5.36 ± 0.08, 2.49 ± 0.06, and 2.04 ± 0.01 ppm). However, after 2 weeks from the injection, the RSC values were less than 10% of the initial retention (0.01 ± 0.01, 0.32 ± 0.01, and 0.01 ± 0.01 ppm). In the TA group, all week 1 values (0.41 ± 0.03, 0.97 ± 0.08, and 0.35 ± 0.02 ppm) and week 2 values (0.94 ± 0.01, 1.18 ± 0.04, Table 1. Residual steroid concentration (RSC) in the extracted spinal segments according to the time-points, drug formulations, and locations. Table 1 presents the raw data of the study results. Values are expressed as mean ± standard deviation, because each sample was analyzed three times for accuracy. To intuitively compare RSCs, the values obtained from animals in each group sacrificed immediately after ESI (Day 1) were used as standard (set 100%, relatively). RSC residual steroid concentration, PLGA poly (lactic-co-glycolic acid), TA triamcinolone acetonide, ESI epidural steroid injection. www.nature.com/scientificreports/ and 1.01 ± 0.02 ppm) were less than 10% of its day 1 RSC value. At week 4, extremely small amount of RSCs were detected in both groups, which were less than 5% of the initial RSC values. When investigating the RSC trend over time, the slope of RSC reduction was milder in the PLGA group than in the TA group during the first week after injection (Fig. 6). Compared to the TA group, the PLGA group had a higher RSC at week 1 despite a low initial steroid retention (106.6%, 49.5%, and 40.6% in PLGA group; 3.2%, 7.5%, and 2.7% in TA group, Table 1). However, at week 2, the PLGA group showed fewer steroid retention than in TA group, although both contained less than 10% of the initial retention dose (0.2%, 6.4%, and 0.2% in PLGA group; 7.2%, 9.1%, and 7.8% in TA group; Table 1). The RSC values differed depending on the groups over time (P < 0.001). The RSC value was significantly higher in the injection segment (L6/L7) than in the adjacent segment (L4/L5) in both groups (P = 0.012).

Injection segment (ppm) Adjacent segment (ppm) Injection segment (ppm) Adjacent segment (ppm)
No gait complications were noted in the animals before euthanization, except for a rabbit in the TA group, which was sacrificed at week 2 with spinal cord injury during the ESI procedure.

Discussion
In this study, the injection segment showed more steroid retention than the adjacent segment in both PLGA and TA group. The amount of steroid retention in the lumbar segments decreased over time in both groups. These trends of steroid retention indicate the validity of our animal model, which was developed in previous studies 30,31 . Steroid retention showed different trends depending on the drug formulation. When using PLGA microspheres, nearly half of the steroids remained in the tissue at first week despite the lower initial retention compared to injection of the steroid itself. However, using PLGA microspheres were associated with less steroid retention after 2 weeks compared to using steroid itself.
Week 1 results of this study are consistent with published studies. Previous studies have reported that small delivery systems allow for slow dissolution of drugs and steady drug-tissue binding in both in vivo and in vitro environments [16][17][18][19][20][21]32 . In particular, the PLGA microspheres showed increased drug retention in the knee and the paraspinal muscles after intra-articular and intramuscular injection [27][28][29][30] . However, in this study, the PLGA microspheres improved steroid retention within the epidural space only during the first week after ESI.
In current study, the use of PLGA microspheres reduced steroid retention at week 2. However, too few steroids remained in both groups to expect any clinical effect. This unexpected result is probably due to leakage from the epidural space, phagocytosis, and biodegradation of the microspheres. The epidural space is anatomically and compositionally different from paraspinal muscle or cartilage of the knee joint used in previous studies. The epidural space is not a confined structure so that injected drugs can spread immediately into the epidural space and can be leaked through neural foramina. In addition, the epidural space is a fat-containing structure rich in lymph and vascular channels that transport macrophages. The injected drug is removed from the body through the phagocytosis of macrophages-the smaller the target particle size, the more active the reaction [33][34][35][36] . Moreover, the maximal phagocytosis occurred in particles with 2-3 um which was used in current study to www.nature.com/scientificreports/ prevent serious leaks expected when using smaller particles and to avoid the embolic infarction expected when using larger particles [35][36][37] . The in vivo biodegradation of PLGA is influenced by tunable factors including physical and biochemical properties of the material such as size, molecular weight, and surface charge. In particular, the degradation rate and release of the drug is generally accelerated as the molecular weight of PLGA decreases 38 . Therefore, in this study, relatively low molecular weight of PLGA may have contributed to its rapid biodegradation, fast drug release, and short-term drug retention in tissues. Nevertheless, the fact that the PLGA reduced steroid retention more slowly during the first week suggests potential for clinical use. In pain management with ESI, it is important to effectively reduce inflammation at an early stage. In the same context, keeping the drug concentration constant above the threshold is important to achieve therapeutic effect. The PLGA has an advantage of allowing higher drug retention at an early phase without sudden dose peaks in vivo with injection. Another benefit may be that the PLGA induces low steroid retention on the day of injection. This is because sudden dose peaks can be associated with systemic side effects of single steroid injections. In addition, the PLGA showed significantly lower plasma level than conventional drugs when the same amount was administered 27 . Therefore, PLGA has the potential to contribute to ESI once an appropriate injection dose is established. Future injection dose should be established to maintain the maximum therapeutic www.nature.com/scientificreports/ dose in the tissue without increasing plasma concentrations. Further studies are needed to determine the injection dose or to optimize the properties of the PLGA formulation-such as particle size, hydrophilic properties, ionic character-for local retention. Some limitations of our experimental study must be acknowledged. First, it may be difficult to generalize our results because we used a small number of rabbits in an experimental design based on the 3R principle (replace, reduce, refine)-designed to generate the maximum amount of knowledge using the smallest possible number of animals. Another limitation is that the incidence of embolic infarction was not monitored except prior to sacrifice, as the experiment focused on determining the topical maintenance efficacy of the new formulation. Further studies would be needed to evaluate the safety and complications associated with the formulation. Third, we used a single size of microspheres and performed only the midline approach to the lumbar spine in all animals. Experimentation with different sized particles or other injection methods such as facet joint or transforaminal approach may give different results. Fourth, histopathologic analysis was not conducted in this study. This is because the focus of the study was to compare the quantity of remaining microspheres depending on the drug formulation. Finally, researchers have tried to inject the drug identically, but the distribution of the drug may vary from individual to individual. For example, in some animals, the injected drug may have been lost immediately through the neural foramen of the spine.
In summary, more steroids remained in the epidural space during the first week after PLGA injection despite the low initial retention compared to conventional drugs. Our findings suggest the potential for PLGA use in ESI if injection doses are established and formulation properties are adjusted. Further research is needed to clarify this point.

Data availability
Data are available on reasonable request.